Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

A transient outward current dependent on external calcium in rat cerebellar granule cells

Summary The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to anI _A-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca²⁺, but significantly inhibited by 3mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED₅₀=0.7mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca²⁺, Mn²⁺ and Mg²⁺. In 2.8mM Mn²⁺, the transient potassium conductance was comparable to that in 2.8mM Ca²⁺, while in 2.8mM Mg²⁺ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca²⁺ was present, Mg²⁺ up to 5mM had no effect. The transient current increased with increasing concentrations of external Ca²⁺, [Ca²⁺] _o , and the maximum conductancevs. [Ca²⁺] _o curve could be approximated by a one-site model. In addition, the current recorded with 5.5mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca²⁺ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.     

Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria

A23187, a new antibiotic with ionophore properties, uncoupled oxidative phosphorylation in mitochondria which oxidized either malate plus glutamate or succinate. Ca²⁺, but not Mg²⁺, enhanced the uncoupling effect. Fluorescence of ANS¹ was increased by A23187 suggesting the mitochondrial membranes were de-energized. This de-energization was presumably by activation of the energy-dependent uptake of Ca²⁺. The steady-state measurements of murexide-divalent cation complexes showed that A23187 caused mitochondria to release the accumulated Ca²⁺ to the medium. This reduced the transmembrane Ca²⁺ gradient even though normal active Ca²⁺ uptake could take place. A23187 inhibited activity of ATPase induced by 2,4-dinitrophenol, valinomycin, and Ca²⁺. The addition of Mg²⁺ could prevent this inhibition presumably by maintaining the endogenous Mg²⁺ concentration. The above metabolic events could be explained by the fact that molecules of A23187 function in the mitochondrial inner membrane as mobile carriers for divalent cations.     

Leucocyte locomotion and chemotaxis : The influence of divalent cations and cation ionophores

Human blood neutrophil leucocytes and monocytes incubated in the absence of Ca²⁺ and Mg²⁺ showed reduced, but still substantial migration into micropore filters towards chemotactic agents, compared with cells migrating in a divalent cation-rich medium. This reduction in migration could be reversed by adding low doses of divalent cation ionophores (X537A or A23187) to the Ca²⁺- and Mg²⁺-free medium which suggests that migrating leucocytes in media depleted of extracellular divalent cations can make use of intracellular divalent cations and that the intracellular cation exchange necessary for locomotion is facilitated by the ionophores. At higher doses, the ionophores inhibited locomotion, as did procaine which reduces membrane permeability to cations. Little effect of K⁺ depletion or of ouabain on leucocyte locomotion was noted.     

Some characteristics of Ca2+ uptake by yeast cells

Summary Experiments were performed to obtain information on: (i) the specific properties of Ca²⁺ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca²⁺ in yeast. These were obtained mainly by considering actual concentrations of Ca²⁺ in the medium after substracting the amounts bound to the cell. Ak _m of 1.9 m and aV _max of 1.2 nmol (100 mg·3 min)^–1 were calculated.The effects of some inhibitors and other cations on Ca²⁺ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca²⁺ uptake in yeast.The effects of Mg²⁺ on the uptake of Ca²⁺ agree with the existence of a single transport system for both divalent cations.The actions of Na⁺ and K⁺ on the transport of Ca²⁺ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.     

ATTEMPTS TO CULTURE ISOLATED URODELAN BLASTOMERES THAT CONTINUE TO DIVIDE WITHOUT AGGREGATING*

The blastomeres isolated from urodelan blastulae continued to divide without aggregating of daughter cells when inoculated with Ca²⁺-free neutral Holtfreter solution into glass culture dishes coated with agar. When standard Holtfreter solution with pH 8.2 was used as a culture medium, Ca²⁺ content from 1/40 to 1/20 of the original strength was essential for the purpose of the present observations; other divalent cations such as Mg²⁺, Ba²⁺ or Mn²⁺ replaced Ca²⁺. Under these experimental conditions, cell pedigrees were obtained during the incubation period. The greatest number of cell divisions so far observed in vitro was 8 for Hynobius lichenatus, and 9 for Cynops pyrrhogaster. Some related observations on the behavior of isolated blastomeres are also presented.     

Identification of a wheat (Triticum aestivum) cell line lacking a specific divalent cation requirement

A wheat (Triticum aestivum L.) cell line, derived from anther culture of an F₁ hybrid, has exogenous Ca²⁺, to that of calcium-dependent cells grown on complete medium. The calcium-independent cell line has been grown in the absence of Ca²⁺ for more than 1.5 years. The cell line grew at a rate similar to that on complete medium for up to 12 weeks, if supplied with any one of the divalent cations, Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Cu²⁺ or Co²⁺, but declined and appeared necrotic when all 6 of these were removed from the medium. The calcium-independence trait, while identified in tissue culture, was also observed in germinated immature embryos of the same hybrid and one of its parental inbred lines.     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.     

Regulation of protein synthesis in rabbit reticulocyte lysates: effect of of Ca2+, Mg2+, and Mn2+ on self-phosphorylation and heterophosphorylation catalyzed by the heme-regulated and double-stranded-RNA-activated protein kinases

The influence of divalent cations Mg²⁺, Mn²⁺, and Ca²⁺ on the cyclic-AMP-independent protein kinases of the heine-regulated and double-stranded-RNA-activated translational inhibitory protein kinases on self-phosphorylation and heterophosphorylation of the substrate (the 38 000-dalton subunit of initiation factor eIF-2) has been examined, Results show that Mg²⁺, Mn²⁺, and Ca²⁺ affect the activities of these enzymes in the following fashion. Mg²⁺ supports both self-phosphorylation and heterophosphorylation efficiently. Mn²⁺ on the other hand supports self-phosphorylation but to a lesser degree the heterophosphorylation, Ca²⁺ promotes neither self-phosphorylation nor hetero-phosphorylation.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.     

The role of divalent cations in interactions between lymphokines and macrophages

The divalent cation requirements of lymphokine-mediated alterations in macrophage function (activation and inhibition of migration) were examined. Normal rabbit alveolar macrophages exposed to incubation supernatants of antigen-stimulated sensitized lymphocytes (lymphokine) were activated, manifested by increased adherence and enhanced bactericidal activity, as compared with control cells. This lymphokine-mediated activation was dependent upon the presence of extracellular Mg²⁺ (but not Ca²⁺). Our data from both current and previous studies suggest that Mg²⁺ influx is necessary for initiation or support of the macrophage activation process. The divalent cation requirements for lymphokine (MIF)-induced inhibition of macrophage migration differed from that of the activation phenomenon. Specifically, both Ca²⁺ and Mg²⁺ were required for expression of MIF activity. Adsorption experiments indicate that these cations are needed for binding of MIF to the macrophage surface.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

Sorbitol Uptake in Plasma Membrane Vesicles Isolated from Immortalized Rabbit TALH Cells: Activation by a Ca2+/Calmodulin-dependent Protein Kinase

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg²⁺, Ca²⁺, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37°C to 4°C. A lower level of uptake was also observed in the presence of 100 μm quinidine and when Ca²⁺ or ATP were omitted from the medium. Membranes preincubated with Mg²⁺, Ca²⁺, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 μm trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca²⁺/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca²⁺/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells. Received: 31 March 1997/Revised: 11 June 1997     

Cations stimulate proton pumping in Catharanthus roseus cells: implication of a redox system?

Abstract During growth, cultured Catharanthus oseus cells produce a transient acidification of the culture medium that may be controlled by cations. The removal of divalent ions from the medium by the chelator EGTA resulted in an inhibition of this acidification. Conversely, acidification can be stimulated by the addition of Ca²⁺, Mg²⁺ and La³⁺ in the basal medium. This acidification process and the proton-linked redox pump previously described (Marigo & Belkoura, 1985) respond in a similar manner to cations. These two systems, which are both inhibited by the Ca²⁺ -calmodulin antagonist calmidazolium, could be regulated by the Ca²⁺-calmodulin complex. By using ionic surfactant (CP⁺, SDS^?) it was demonstrated that the net surface charge of the plasmalemma plays a role in the activation of the two pumping processes. These results are interpreted to indicate that a transmembrane redox system could provide the energy for electrogenic proton extrusion.     

Novel function of eubacterial flagella: role in aggregation of a marine bacterium

Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN₃. Aggregates were not observed when Mg²⁺ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca²⁺, Mn²⁺, and Ba²⁺ could replace Mg²⁺. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs⁺ or Li⁺ instead of Mg²⁺. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.Abbreviation ASW artificial seawater     

Divalent cations regulate the organization of integrins αvβ3 and αvβ5 on the cell surface

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins αvβ3 and αvβ5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin αvβ3 organized in focal contacts when Mn²⁺ or Mg²⁺ was present, but not in Ca²⁺. In contrast, αvβ5 organized in focal contacts only when Ca²⁺ or Mg²⁺ was present. Integrin αvβ5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn²⁺. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites. © 1996 Wiley-Liss, Inc.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Correlation between the Suppression of Glucose and Phosphate Uptake and the Release of Protein from Viable Carrot Root Cells Treated with Monovalent Cations

Treating carrot (Daucus carota L.) discs with ice-cold NaCl solutions for 30 minutes caused three effects that appear to be functionally related: the exchange of tissue Ca²⁺ and Mg²⁺ for Na⁺, the release of protein, and the suppression of active uptake of glucose and orthophosphate. Cyclosis continued apparently unabated after treatment with NaCl at concentrations of up to 0.25 m, so the cells remained viable and energetically competent. The correlation between the release of Ca²⁺ and Mg²⁺ and release of protein, and between these effects and the suppression of glucose and orthophosphate uptake, supports the hypothesis that divalent cations maintain, and monovalent cations disrupt, linkages between the outer cell surface and proteins required for active solute uptake. Calcium preserved uptake activity only when it was added in time to prevent the release of protein. Cells gradually recovered some glucose uptake activity after it had been completely inactivated by treatment with 0.25 m NaCl. This recovery occurred in the absence of added Ca²⁺. It was inhibited by puromycin and so appears to require some protein synthesis. Beet (Beta vulgaris L.) discs were more resistant than carrot discs to treatment with NaCl solutions, thus reflecting the difference in tolerance of the two species to sodicity.